Reductive alkylation of glycopeptide antibiotics

ABSTRACT

This invention is concerned with improved processes for reductive alkylation of glycopeptide antibiotics. The improvement residing in providing a source of copper which results in the initial production of a copper complex of the glycopeptide antibiotic. Reductive alkylation of this complex favors regioselective alkylation and increased yields. Copper complexes of the glycopeptide antibiotic starting materials and of the alkylated products are also part of the invention.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/031,596, filed Nov. 21, 1996.

BRIEF SUMMARY

The present invention is directed to improved methods for reductivelyalkylating glycopeptide antibiotics. The invention provides increasedregioselectivity of reaction among multiple sites and thereby results inincreased yields of the preferred product. In particular, the inventionis directed to methods for preferentially conducting a reductivealkylation reaction on an amine on the saccharide of a glycopeptideantibiotic having one or more additional amines.

The essence of the invention is the discovery that conducting thereaction in the presence of soluble copper favors preferential reactionwith the amine on the saccharide position, and thereby improves theyields of the reductive alkylation at this site. The initial step is theformation of a copper complex of the glycopeptide antibiotic, whichsubsequently undergoes the reductive alkylation. This invention is alsodirected to these copper complexes of the starting glycopeptideantibiotics. The alkylated glycopeptide antibiotic products are obtainedas copper complexes, which are another embodiment of the presentinvention.

DETAILED DESCRIPTION

The present invention relates to reductive alkylation of glycopeptideantibiotics.

The glycopeptide antibiotics are a large class of substances eitherproduced by microorganisms, or produced by microorganisms and thereaftersubsequently modified in part. Two of these, vancomycin and teicoplanin,are sold as antibacterial products, but many others have been discoveredand are being considered for development, especially since the emergencein the late 1980s of resistance to various antibiotics, including theglycopeptides themselves. The entire class of glycopeptide antibioticsis well described in "Glycopeptide Antibiotics", edited by RamakrishnanNagarajan (Marcel Dekker, Inc., New York, 1994). Among the more recentlydiscovered glycopeptides are those known as A82846A (also calledereomomycin), A82846B (also known as chloroorienticin A), A82846C (alsoknown as orienticin C), and orienticin A. The present invention ispreferred for use with vancomycin type glycopeptide antibiotics,including vancomycin, A82846A, A82846B, A82846C, and orienticin A; theinvention is especially preferred for use with A82846B.

Many modifications of naturally-occurring glycopeptides have been made.Among the modifications are reductive alkylations of reactive amine(s)in glycopeptides. See, for example, U.S. Pat. No. 4,698,327 describingreductive alkylations of vancomycin, and EPO 435 503 A1 and EPO 667 353A1, both of which describe reductive alkylations of a variety ofglycopeptides including vancomycin, A82846A, A82846B, A82846C, andorienticin A. These references describe reductive alkylations whichintroduce into the parent glycopeptides a great variety of alkyl groups.

U.S. Pat. No. 4,698,327 describes alkylated vancomycin compounds of theformula: ##STR1## wherein

R is hydrogen or methyl;

n is 1 or 2; and

R₁ is hydrogen or methyl;

R₂ and R₃, independently, are hydrogen or a group of the formula: R₆ R₇CH--;

R₆ and R₇ are independently R₅, R₅ -(C₁ -C₅ -alkyl) or R₅ -(C₂ -C₅-alkenyl);

R₅ is hydrogen, C₁ -C₁₀ -alkyl, C₂ -C₁₀ -alkenyl, C₁ -C₄ alkoxy, C₃ -C₁₀-cycloalkyl, C₅ -C₁₂ -cycloalkenyl, phenyl, naphthyl, indenyl,tetralinyl, decalinyl, adamantyl, a monocyclic heterocyclic ring systemcomprising 3 to 8 atoms in the ring or a bicyclic heterocyclic ringsystem comprising 6 to 11 atoms, provided that at least one atom of thering system is carbon and at least one atom of the ring system is aheteroatom selected from O, N, and S, and R₅ may be substituted with oneor more hydroxy, nitro, C₁ -C₁₀ -alkoxy, C₁ -C₁₀ -alkyl, phenyl, C₁ -C₆-alkylthio, nitrile, halo, C₂ -C₄ -acylamino, amino, C₁ -C₄-dialkylamino groups; and R₄ is hydrogen, provided that: (1) at leastone of R₂ and R₃ must be other than hydrogen; (2) when n is 2, R must behydrogen; (3) when R is methyl and R₃ is hydrogen, R₂ cannot be methyland (4) when R and R₁ are both methyl, then R₂ is hydrogen or methyl andn is 1.

EPO 435 503 A1 is directed to alkylated and acylated glycopeptides ofthe formula: ##STR2## wherein:

R is hydrogen or a (4-epi-vancosaminyl)--O--glucosyl group of formula##STR3## or the glucosyl group of formula ##STR4##

X is hydrogen or chloro;

Y is hydrogen or chloro;

R₁, R₂, and R₃ are independently hydrogen; C₁ -C₁₂ alkyl; C₂ -C₉alkanoyl; or a group of formula ##STR5##

n is 1 to 3;

R₄ is hydrogen, halo, C₁ -C₈ alkyl, C₁ -C₈ alkoxy, or a group of formula##STR6##

R₅ and R₆ are independently hydrogen or C₁ -C₃ alkyl;

p is 0 to 2;

m is 2 or 3, and r=3-m; provided that, where R is a(4-epi-vancosaminyl)--O--glucosyl group, R₁, R₂, and R₃ are not allhydrogen, and where R is hydrogen or a glucosyl group, R₁ and R₃ are notboth hydrogen.

Where R is (4-epi-vancosaminyl)--O--glucosyl, the glycopeptides sodefined are

X=H, Y=Cl, A82846A

X=y=Cl, A82846B

X=Y=H, A82846C

X=Cl, Y=H, orienticin A.

Thus, EPO 435 503 A1 describes alkyl derivatives of A82846A, A82846B,A82846C, and orienticin A wherein the alkyl group is ##STR7## Preferredgroups are C₈ -C₁₂ alkyl and groups of the formula ##STR8## wherein R₄is hydrogen, halo, C₁ -C₈ alkyl, or C₁ -C₈ alkoxy.

EPO 667 353 A1 describes alkylated glycopeptide antibiotics of theformula ##STR9## wherein:

X and Y are each independently hydrogen or chloro;

R is hydrogen, 4-epi-vancosaminyl, actinosaminyl, or ristosaminyl;

R¹ is hydrogen, or mannose;

R² is --NH², --NHCH³, or --N(CH₃)₂ ;

R³ is --CH₂ CH(CH₃)₂, p--OH, m--Cl!phenyl, p-rhamnose-phenyl, orp-rhamnose-galactose!phenyl, p-galactose-galactose!phenyl, p--CH₃O--rhamnose!phenyl;

R⁴ is --CH₂ (CO)NH₂, benzyl, p--OH!phenyl, or p--OH, m--Cl!phenyl;

R⁵ is hydrogen, or mannose;

R⁶ is vancosaminyl, 4-epi-vancosaminyl, L-acosaminyl, L-ristosaminyl, orL-actinosaminyl;

R⁷ is (C₂ -C₁₆)alkenyl, (C₂ -C₁₂)alkynyl, (C₁ -C₁₂ alkyl)-R₈, (C₁ -C₁₂alkyl)-halo, (C₂ -C₆ alkenyl)-R₈, (C₂ -C₆ alkynyl)-R₈, (C₁ -C₁₂alkyl)--O--R₈, and is attached to the amino group of R⁶ ;

R⁸ is selected from the group consisting of:

a) multicyclic aryl unsubstituted or substituted with one or moresubstituents independently selected from the group consisting of:

(i) hydroxy,

(ii) halo,

(iii) nitro,

(iv) (C₁ -C₆)alkyl,

(v) (C₂ -C₆)alkenyl,

(vi) (C₂ -C₆)alkynyl,

(vii) (C₁ -C₆)alkoxy,

(viii) halo-(C₁ -C₆)alkyl,

(ix) halo-(C₁ -C₆)alkoxy,

(x) carbo-(C₁ -C₆)alkoxy,

(xi) carbobenzyloxy,

(xii) carbobenzyloxy substituted with (C₁ -C₆)alkyl, (C₁ -C₆)alkoxy,halo, or nitro,

(xiii) a group of the formula --S(O)_(n') --R⁹, wherein n' is 0-2 and R⁹is (C₁ -C₆)alkyl, phenyl, or phenyl substituted with (C₁ -C₆)alkyl, (C₁-C₆)alkoxy, halo, or nitro, and

(xiv) a group of the formula --C(O)N(R¹⁰)₂ wherein each R¹⁰ substituentis independently hydrogen, (C₁ -C₆)-alkyl, (C₁ -C₆)-alkoxy, phenyl, orphenyl substituted with (C₁ -C₆)-alkyl, (C₁ -C₆)-alkoxy, halo, or nitro;

b) heteroaryl unsubstituted or substituted with one or more substituentsindependently selected from the group consisting of:

(i) halo,

(ii) (C₁ -C₆)alkyl,

(iii) (C₁ -C₆)alkoxy,

(iv) halo-(C₁ -C₆)alkyl,

(v) halo-(C₁ -C₆)alkoxy,

(vi) phenyl,

(vii) thiophenyl,

(viii) phenyl substituted with halo, (C₁ -C₆)alkyl, (C₂ -C₆)alkenyl, (C₂-C₆)alkynyl, (C₁ -C₆)alkoxy, or nitro,

(ix) carbo-(C₁ -C₆)alkoxy,

(x) carbobenzyloxy,

(xi) carbobenzyloxy substituted with (C₁ -C₆)alkyl, (C₁ -C₆) alkoxy,halo, or nitro,

(xii) a group of the formula --S(O)_(n') --R⁹, as defined above,

(xiii) a group of the formula --C(O)N(R¹⁰)₂ as defined above, and

(xiv) thienyl;

c) a group of the formula: ##STR10##

wherein A¹ is --OC(A²)₂ --C(A²)₂ --O--, --O--C(A²)₂ --O--, --C(A²)₂--O--, or --C(A²)₂ --C(A²)₂ --C(A²)₂ --C(A²)₂ --, and each A²substituent is independently selected from hydrogen, (C₁ -C₆)-alkyl, (C₁-C₆)alkoxy, and (C₄ -C₁₀)cycloalkyl;

d) a group of the formula: ##STR11##

wherein p is from 1 to 5; and

R¹¹ is independently selected from the group consisting of:

(i) hydrogen,

(ii) nitro,

(iii) hydroxy,

(iv) halo,

(v) (C₁ -C₈)alkyl,

(vi) (C₁ -C₈)alkoxy,

(vii) (C₉ -C₁₂)alkyl,

(viii)(C₂ -C₉)alkynyl,

(ix) (C₉ -C₁₂)alkoxy,

(x) (C₁ -C₃)alkoxy substituted with (C₁ -C₃)alkoxy, hydroxy, halo(C₁-C₃)alkoxy, or (C₁ -C₄)alkylthio,

(xi) (C₂ -C₅)alkenyloxy,

(xii) (C₂ -C₁₃) alkynyloxy

(xiii) halo-(C₁ -C₆) alkyl,

(xiv) halo-(C₁ -C₆)alkoxy,

(xv) (C₂ -C₆)alkylthio,

(xvi) (C₂ -C₁₀)alkanoyloxy,

(xvii) carboxy-(C₂ -C₄)alkenyl,

(xviii) (C₁ -C₃)alkylsulfonyloxy,

(xix) carboxy-(C₁ -C₃)alkyl,

(xx) N- di(C₁ -C₃)-alkyl!amino-(C₁ -C₃)alkoxy,

(xxi) cyano-(C₁ -C₆)alkoxy, and

(xxii) diphenyl-(C₁ -C₆)alkyl,

with the proviso that when R¹¹ is (C₁ -C₈)alkyl, (C₁ -C₈)alkoxy, orhalo, p must be greater or equal to 2, or when R⁷ is (C₁ -C₃ alkyl)-R⁸then R¹¹ is not hydrogen, (C₁ -C₈)alkyl, (C₁ -C₈)alkoxy, or halo;

e) a group of the formula: ##STR12##

wherein q is 0 to 4;

R¹² is independently selected from the group consisting of:

(i) halo,

(ii) nitro,

(iii) (C₁ -C₆)alkyl,

(iv) (C₁ -C₆)alkoxy,

(v) halo-(C₁ -C₆)alkyl,

(vi) halo-(C₁ -C₆)alkoxy, and

(vii) hydroxy, and

(vii) (C₁ -C₆)thioalkyl;

r is 1 to 5; provided that the sum of q and r is no greater than 5;

Z is selected from the group consisting of:

(i) a single bond,

(ii) divalent (C₁ -C₆)alkyl unsubstituted or substituted with hydroxy,(C₁ -C₆)alkyl, or (C₁ -C₆)alkoxy,

(iii) divalent (C₂ -C₆)alkenyl,

(iv) divalent (C₂ -C₆)alkynyl, or

(v) a group of the formula --(C(R¹⁴)₂)_(s) --R¹⁵ -- or --R¹⁵--(C(R¹⁴)₂)_(s) --, wherein s is 0-6; wherein each R¹⁴ substituent isindependently selected from hydrogen, (C₁ -C₆)-alkyl, or (C₄ -C₁₀)cycloalkyl; and R¹⁵ is selected from --O--, --S--, --SO--, --SO₂ --,--SO₂ --O--, --C(O)--, --OC(O)--, --C(O)O--, --NH--, --N(C₁ -C₆alkyl)--, and --C(O)NH--, --NHC(O)--, N═N;

R¹³ is independently selected from the group consisting of:

(i) (C₄ -C₁₀)heterocyclyl,

(ii) heteroaryl,

(iii) (C₄ -C₁₀)cycloalkyl unsubstituted or substituted with (C₁-C₆)alkyl, or

(iv) phenyl unsubstituted or substituted with 1 to 5 substituentsindependently selected from: halo, hydroxy, nitro, (C₁ -C₁₀) alkyl, (C₁-C₁₀) alkoxy, halo-(C₁ -C₃) alkoxy, halo-(C₁ -C₃) alkyl, (C₁ -C₃)alkoxyphenyl, phenyl, phenyl-(C₁ -C₃)alkyl, (C₁ -C₆)alkoxyphenyl,phenyl-(C₂ -C₃)alkynyl, and (C₁ -C₆)alkylphenyl;

f) (C₄ -C₁₀)cycloalkyl unsubstituted or substituted with one or moresubstituents independently selected from the group consisting of:

(i) (C₁ -C₆)alkyl,

(ii) (C₁ -C₆)alkoxy,

(iii) (C₂ -C₆)alkenyl,

(iv) (C₂ -C₆)alkynyl,

(v) (C₄ -C₁₀) cycloalkyl,

(vi) phenyl,

(vii) phenylthio,

(viii) phenyl substituted by nitro, halo, (C₁ -C₆)alkanoyloxy, orcarbocycloalkoxy, and

(ix) a group represented by the formula --Z--R¹³ wherein Z and R¹³ areas defined above; and

g) a group of the formula: ##STR13## wherein

A³ and A⁴ are each independently selected from

(i) a bond,

(ii) --O--,

(iii) --S(O)_(t) --, wherein t is 0 to 2,

(iv) --C(R¹⁷)₂ --, wherein each R¹⁷ substituent is independentlyselected from hydrogen, (C₁ -C₆)alkyl, hydroxy, (C₁ -C₆)alkyl, (C₁-C₆)alkoxy, or both R¹⁷ substituents taken together are O,

(v) --N(R¹⁸)₂ --, wherein each R₁₈ substituent is independently selectedfrom hydrogen; (C₁ -C₆)alkyl; (C₂ -C₆)alkenyl; (C₂ -C₆)alkynyl; (C₄-C₁₀)cycloalkyl; phenyl; phenyl substituted by nitro, halo, (C₁-C₆)alkanoyloxy; or both R¹⁸ substituents taken together are (C₄-C₁₀)cycloalkyl;

R¹⁶ is R¹² or R¹³ as defined above; and

u is 0-4.

In this reference, preferred glycopeptide antibiotics are A82846A,A82846B, A82846C, and orienticin A; preferred alkyls are those whereinR⁷ is CH₂ -R₈ ; and preferred R⁸ moieties are those defined as groups"(d)" and "(e)".

The present invention can be utilized to make the alkylatedglycopeptides described in these references. Preferred alkylatedglycopeptides which can be prepared by the present process include thefollowing:

N⁴ -n-octylA82846B

N⁴ -n-decylA82846B

N⁴ -benzylA82846B

N⁴ -(p-chlorobenzyl)A82846B

N⁴ -(p-bromobenzyl)A82846B

N⁴ -(p-propylbenzyl)A82846B

N⁴ -(p-isopropylbenzyl)A82846B

N⁴ -(p-butylbenzyl)A82846B

N⁴ -(p-isobutylbenzyl) A82846B

N⁴ -(p-pentylbenzyl)A82846B

N⁴ -(p-isohexylbenzyl)A82846B

N⁴ -(p-octylbenzyl)A82846B

N⁴ -(p-propoxybenzyl)A82846B

N⁴ -(p-isopropoxybenzyl)A82846B

N⁴ -(p-butoxybenzyl) A82846B

N⁴ -(p-tert-butoxybenzyl)A82846B

N⁴ -(p-pentyloxybenzyl)A82846B

N⁴ -(p-hexyloxybenzyl)A82846B

N⁴ -(o-hexyloxybenzyl)A82846B

N⁴ -(p-heptyloxybenzyl)A82846B

N⁴ -(p-octyloxybenzyl)A82846B

N⁴ -phenethylA82846B

N⁴ -(4-phenylbenzyl)A82846B

N⁴ -(4-(4-chlorophenyl)benzylA82846B

N⁴ -(4-(4-methylbenzyloxy)benzyl)A82846B

N⁴ -(4-(4-ethylbenzyloxy)benzyl)A82846B

N⁴ -(4-(4-chlorophenethyl)benzyl)A82846B

N⁴ -(4-(2-(4-methoxyphenyl)ethynyl)benzyl)A82846B.

The references noted above describe the reductive alkylation ascomprising a first step, in which the glycopeptide is reacted with therespective aldehyde or ketone to form a Schiff's base, which in a secondstep is reduced to the desired alkylated product. In one variation ofthis procedure, EPO 667 353 A1 describes a process in which the reducingagent is added simultaneously with the glycopeptide and aldehyde orketone. The references say that any chemical reducing agent can beemployed, but the references also suggest a preference for sodiumcyanoborohydride.

Essentially all glycopeptides contain multiple reactive sites.Manipulation of these multiple sites is not uniformly advantageous. Itis sometimes desired to react the glycopeptide regioselectivity, to havethe reaction occur at only one of multiple sites. This is equally truein the case of reductive alkylations of glycopeptides. An example ofthis is A82846B. While derivatives alkylated on the leucine amine (N¹)and/or the monosaccharide (N⁶) are active as antibacterials, alkylationof the N⁴ (disaccharide) amine appears to be preferred. Pharmaceuticalpractices require a relatively pure form, and therefore preferentialreaction of the N⁴ site is desirable in order to achieve a highly pureN⁴ -alkylated product.

The present invention provides a technique for obtaining reactionpreferentially on the amine on a saccharide at the N⁴ position in theglycopeptide antibiotic. In the case of vancomycin, A82846A, A82846B,A82846C, and orienticin A, the present process reduces reactivity atsites N¹ and N⁶ and thereby increases reaction selectivity for the N⁴(disaccharide) site. The invention requires the initial preparation of asoluble copper complex of the glycopeptide, which is then reductivelyalkylated. The soluble copper complex is achieved by reacting theglycopeptide antibiotic with copper, typically by adding a source ofsoluble copper to a reaction mixture containing the glycopeptideantibiotic. The identity of the copper source is not critical, so longas it is at least partially soluble and does not negatively impact thepH. Such a copper salt can be used in anhydrous or hydrated form. Apreferred source of copper is copper (II) acetate, most convenientlyemployed as the hydrate.

Supplying copper to the reaction mixture results in the initialproduction of a copper complex with the glycopeptide antibiotic startingmaterial, typically in a 1:1 ratio. This copper complex of theglycopeptide antibiotic starting material is one of the features of thepresent invention.

The reducing agent to be employed in the present invention is sodiumcyanoborohydride or pyridine•borane complex.

The identity of the solvent is important. Straight methanol has givenhigh yields, and it is expected that methanol somewhat diluted as withDMF or DMSO would provide acceptable yields. Other solvents have notproduced satisfactory results. Therefore, the reaction solvent is atleast predominantly methanol.

The reaction should be conducted at a pH of 6-8, and preferably at a pHof 6.3-7.0.

The amounts of reactants and reagents to be employed are not critical;amounts to maximize the yield of product will vary somewhat with theidentity of the reactants. The reaction consumes the glycopeptideantibiotic and the aldehyde or ketone in equimolar amounts. A slightexcess of the aldehyde or ketone, e.g., 1.3 to 1.7:1, is preferred. Theamount of the glycopeptide antibiotic to be used must be corrected forits purity. The reaction consumes an equimolar amount of the reducingagent. At least that amount should be employed, and a slight excess ispreferred. The amount of soluble copper is not critical when employingsodium cyanoborohydride as reducing agent. When employingpyridine•borane as reducing agent, the amount of soluble copper to beemployed is more important, since excess copper will react with thepyridine•borane. Regardless of the identity of the reducing agent, thepresent process first results in the formation of a 1:1 complex with theglycopeptide antibiotic; therefore, the copper is preferably present inan amount approximately equimolar with the glycopeptide antibiotic.Amounts exceeding one molar equivalent (in the case of pyridine•borane)or two molar equivalents (in the case of sodium cyanoborohydride) areundesirable.

Summarizing the foregoing, the ideal amounts to be employed are a ratioof:

glycopeptide:aldehyde or ketone:reducing agent:copper salt of:

1:1.3 to 1.5:1.3:1

with the exception that when using pyridine•borane complex as reducingagent, the preferred ratio is:

1:1.3 to 1.7:1.5:0.9 to 1.0.

The concentration of the reactants in the solvent has some bearing onthe process. Methanol volume relative to mass of glycopeptide antibioticcan vary from 50:1 to 500:1; a 100:1 dilution appears to be a useful,practical ratio, although higher dilutions may give slightly higheryields.

The temperature at which the process is carried out is not critical.Reaction mixtures in methanol boil at about 67° C., thereby setting themaximum temperature when employing straight methanol as the solvent.Higher temperatures are of course possible when employing mixtures ofmethanol or when operating under pressure. Lower temperatures can betolerated, but preferably not lower than about 45° C. The idealcondition for sodium cyanoborohydride as reducing agent is the use ofstraight methanol and conducting the reaction at reflux; the idealcondition for pyridine•borane as reducing agent is also the use ofstraight methanol but at temperatures of about 58-63° C.

Some product is produced with even short reaction times. Longer reactiontimes, such as from 6 hours to 48 hours, are preferred. However, theideal reaction time appears to be approximately 20 to 25 hours. Longertimes may increase the yield of products alkylated at undesired sites inthe glycopeptide antibiotic.

In carrying out the present invention, the glycopeptide antibiotic andcopper are preferably mixed in a solvent, creating the soluble coppercomplex of the glycopeptide antibiotic, and the aldehyde and reducingagents are then added. However, the precise order of addition is notcritical. Portionwise addition of the reducing agent is preferred, andis required for good results when employing pyridine•borane complex asreducing agent. The reaction is continued for a period of time, afterwhich the product is produced and can be separated from the reactionmixture.

Upon the completion of the reaction period, the reaction mixture ispreferably quenched, as by the addition of sodium borohydride. Thisreagent consumes residual aldehyde or ketone and thereby preventsfurther undesired reactions.

The product is isolated from the reaction mixture as a copper complex ofthe alkylated glycopeptide. Isolation is achieved by concentration ofthe reaction mixture and precipitation of the complex by addition of anantisolvent such as ethyl acetate, acetone, 1-propanol, isopropylalcohol, or preferably acetonitrile. The complex can be broken byaqueous treatment at pH ² 4, freeing the simple alkylated glycopeptideproduct, which can, if desired, be purified in conventional manner.

The following examples illustrate the present invention and will enablethose skilled in the art to practice the same.

REFERENCE EXAMPLE A (NO COPPER)

A82846B (6.0 g, 76.5% potency, 4.59 bg, 2.88 mmol),4'-chloro-4-biphenylcarboxaldehyde (0.86 g, 3.97 mmol), and sodiumcyanoborohydride (84 mg, 1.34 mmol) were added to 600 mL methanol andthe solution was heated at reflux for 3 hours. An additional portion ofsodium cyanoborohydride (84 mg, 1.34 mmol) was added and the mixture washeated 3 hours longer at reflux. A final portion of sodiumcyanoborohydride (84 mg, 1.34 mmol) was added and heating at reflux wascontinued an additional 17 hours. The clear colorless solution wascooled to ambient temperature and concentrated to 130 mL on a rotaryevaporator. The product was precipitated by addition of 200 mL ofisopropyl alcohol over 2 hours. After cooling to 0° C. and stirring 1hour, filtration afforded N⁴ -(4-(4-chlorophenyl)benzyl)A82846B as awhite solid (5.61 g, 49.3% potency, 2.77 bg, 53.7%).

EXAMPLE 1

A82846B(0.50 g, 76.3% potency, 0.38 bg, 0.24 mmol),4'-chloro-4-biphenylcarboxaldehyde (70 mg, 0.32 mmol), and cupricacetate monohydrate (51 mg, 0.26 mmol) were stirred in 50 mL methanol.Sodium cyanoborohydride (20 mg, 0.32 mmol) was added and the solutionwas heated at reflux for 23 hours. The clear purple solution was cooledto ambient temperature and 12% sodium borohydride in aqueous 14 M sodiumhydroxide (0.03 mL, 0.14 mmol) was added. One drop of acetic acid wasadded to pH adjust the solution to 7.3. An additional portion of 12%sodium borohydride in aqueous 14 M sodium hydroxide (0.23 mL, 0.10 mmol)was added followed by one drop of acetic acid to maintain the solutionpH at 7.3. The mixture was stirred at ambient temperature for 1 hour andconcentrated to 12 mL on a rotary evaporator. The product wasprecipitated by addition of 25 mL of acetonitrile over 20 min. Afterstirring 20 min at ambient temperature, filtration afforded the coppercomplex of N⁴ -(4-(4-chlorophenyl)benzyl)A82846B as a purple solid (0.58g, potency 59.5%, 0.35 bg, 80.3%).

EXAMPLE 2

A82846B (6.0 g, 78.4% potency, 4.7 bg, 2.95 mmol), was stirred in 600 mLmethanol and cupric acetate (0.66 g, 3.6 mmol) was added. After stirringat ambient temperature for 15 min, 4'-chloro-4-biphenylcarboxaldehyde(0.95 g, 4.4 mmol), and sodium cyanoborohydride (0.27 g, 4.3 mmol) wereadded and the mixture was heated at reflux for 24 hours. After coolingto ambient temperature, HPLC analysis of a reaction aliquot afforded ayield of 4.52 g (85.4%) of N⁴ -(4-(4-chlorophenyl)benzyl)A82846B.

EXAMPLE 3

A82846B (2.5 g, 78.5% potency, 1.96 bg, 1.23 mmol), was stirred in 250mL methanol and cupric acetate monohydrate (0.26 g, 1.32 mmol) wasadded. After stirring at ambient temperature for 10 min,4'-chloro-4-biphenylcarboxaldehyde (0.35 g, 1.6 mmol), and sodiumcyanoborohydride (34 mg, 0.54 mmol) were added and the mixture washeated at reflux for 3 hours. An additional portion of sodiumcyanoborohydride (34 mg, 0.54 mmol) was added and the mixture was heated3 hours longer at reflux. A final portion of sodium cyanoborohydride (34mg, 0.54 mmol) was added and heating at reflux continued an additional17 hours. The mixture was cooled to ambient temperature and 12% sodiumborohydride in aqueous 14 M sodium hydroxide (0.14 mL, 0.63 mmol) wasadded. A few drops of acetic acid were added to pH adjust the solutionto 7.3. A second portion of 12% sodium borohydride in aqueous 14 Msodium hydroxide (0.13 mL, 0.6 mmol) was added and a few drops of aceticacid were added to adjust the solution pH to 8.1. After stirring atambient temperature for 2 hours, the reaction mixture was concentratedto 60 mL on a rotary evaporator. Isopropyl alcohol (175 mL) was addeddropwise over a period of 1 hour to precipitate the copper complex of N⁴-(4-(4-chlorophenyl)benzyl)A82846B. Filtration afforded the complex as apurple solid (6.50 g, 26.9% potency as wet cake, 1.75 bg, 79.1%).

EXAMPLE 4

A82846B (2.5 g, 78.5% potency, 1.96 bg, 1.23 mmol), was stirred in 250mL methanol and cupric acetate monohydrate (0.26 g, 1.32 mmol) wasadded. After stirring at ambient temperature for 10 min,4'-chloro-4-biphenylcarboxaldehyde (0.35 g, 1.6 mmol), and sodiumcyanoborohydride (34 mg, 0.54 mmol) were added and the mixture washeated at reflux for 3 hours. An additional portion of sodiumcyanoborohydride (34 mg, 0.54 mmol) was added and the mixture was heated3 hours longer at reflux. A final portion of sodium cyanoborohydride (34mg, 0.54 mmol) was added and heating at reflux continued an additional17 hours. The mixture was cooled to ambient temperature and 12% sodiumborohydride in aqueous 14 M sodium hydroxide (0.14 mL, 0.63 mmol) wasadded. A few drops of acetic acid were added to pH adjust the solutionto 7.3. A second portion of 12% sodium borohydride in aqueous 14 Msodium hydroxide (0.13 mL, 0.6 mmol) was added and a few drops of aceticacid were added to adjust the solution pH to 8.2. After stirring atambient temperature for 1.5 hours, the reaction mixture was concentratedto 60 mL on a rotary evaporator. Isopropyl alcohol (175 mL) was addeddropwise over a period of 1 hour to precipitate the product. It wasfiltered and dried in vacuo to afford the copper complex of N⁴-(4-(4-chlorophenyl)benzyl)A82846B as a purple solid (2.56 g, 62.9%potency, 1.61 bg, 72.9%).

EXAMPLE 5

A82846B (6.0 g, 76.1% potency, 4.56 bg, 2.9 mmol), was stirred in 600 mLmethanol and cupric acetate monohydrate (0.63 g, 3.15 mmol) was added.After stirring at ambient temperature for 15 min,4'-chloro-4-biphenylcarboxaldehyde (0.85 g, 3.9 mmol), and sodiumcyanoborohydride (84 mg, 1.3 mmol) were added and the mixture was heatedat reflux for 3 hours. An additional portion of sodium cyanoborohydride(84 mg, 1.3 mmol) was added and the mixture was heated 3 hours longer atreflux. A final portion of sodium cyanoborohydride (84 mg, 1.3 mmol) wasadded and heating at reflux continued an additional 16 hours. Themixture was cooled to ambient temperature and 50% aqueous sodiumhydroxide solution was added to adjust the pH of the reaction mixture to7.6. Sodium borohydride (0.11 g, 2.9 mmol) was added and the solutionwas stirred 3.5 hours at ambient temperature. The reaction mixture wasconcentrated to 110 mL on a rotary evaporator and isopropyl alcohol (250mL) was added dropwise over a period of 4 hours to precipitate theproduct. After cooling the purple slurry to 0° C. for 1 hour, filtrationafforded the purple complex of N⁴ -(4-(4-chlorophenyl)benzyl)A82846B(11.03 g, 36.2% potency as wet cake, 3.99 bg, 77.6%.

REFERENCE EXAMPLE B (NO COPPER)

A82846B (0.50 q, 84.3% potency, 0.42 bg, 0.26 mmol) was stirred in 50 mLmethanol and 4'-chloro 4-biphenylcarboxaldehyde (72 mg, 0.33 mmol) andpyridine•borane complex (0.033 mL, 0.33 mmol) were added. The mixturewas heated at reflux for 6 hours before being cooled to ambienttemperature. HPLC analysis of a reaction aliquot afforded a yield of0.25 g (53.2%) of N⁴ -(4-(4-chlorophenyl)benzyl)A82846B.

EXAMPLE 6

A82846B (0.50 g, 84.3% potency, 0.42 bg, 0.26 mmol was stirred in 50 mLmethanol and cupric acetate (45 mg, 0.25 mmol) was added. After stirringat ambient temperature for 10 min, 4'-chloro-4-biphenylcarboxaldehyde(84 mg, 0.39 mmol) and pyridine•borane complex (0.039 mL, 0.39 mmol)were added. The mixture was heated at 57° C. for 24 hours before beingcooled to ambient temperature. HPLC analysis of a reaction aliquotafforded a yield of 0.34 g (72.3%) of N⁴-(4-(4-chlorophenyl)benzyl)A82846B.

EXAMPLE 7

A82846B (0.50 g, 76.3% potency, 0.38 bg, 0.24 mmol and cupric acetatemonohydrate (43 mg, 0.216 mmol) were stirred in 50 mL methanol and4'-chloro-4-biphenylcarbox-aldehyde (84.5 mg, 0.39 mmol) andpyridine•borane complex (0.011 mL, 0.11 mmol) were added. The mixturewas heated at 63° C. for 2 hours and an additional portion ofpyridine•borane was added (0.01 mL, 0.1 mmol). After 2 hours more at 63°C. a third portion of pyridine•borane (0.005 mL, 0.05 mmol) was added. Afourth portion of pyridine•borane (0.005 mL, 0.05 mmol) was added 2hours later followed by a fifth portion of pyridine•borane (0.005 mL,0.05 mmol) after another 5 hours at 63° C. The mixture was heated at 63°C. for another 11 hours before being cooled to ambient temperature. HPLCanalysis of a reaction aliquot afforded a yield of 0.34 g (79.2%) of N⁴-(4-(4-chlorophenyl)benzyl)A82846B.

The reactions reported in Reference Examples A and B and Examples 1-7were also evaluated (1) for the amount of the remaining startingglycopeptide, (2) for the amount of products alkylated on amine sitesother than the N⁴ -position, and (3) for the amount ofmultiply-alkylated products. The results are set forth in the followingtable and are expressed as a percentage relative to the intended productmonoalkylated on the N⁴ -amine; yields of the intended product areactual yields as recited in the foregoing examples.

                                      TABLE I    __________________________________________________________________________                            % Di-                                 % Di-        % Mono-   % Mono-                       % Mono-                            alkylated                                 alkylated    Ex. alkylated             %    alkylated                       alkylated                            at both                                 at both                                      % Tri-    No. at N.sup.4             A82846B                  at N.sup.6                       at N.sup.1                            N.sup.4 and N.sup.6                                 N.sup.1 and N.sup.4                                      alkylated    __________________________________________________________________________    RE A        53.7 14.1 3.5  2.4  24.4 15.9 3.4    1   80.3 7.6  1.0  0.4  9.7  5.6  0.7    2   85.4 13.0 2.4  0.7  8.2  5.9  0.9    3   79.1 9.8  1.1  0.5  8.1  6.4  0.6    4   72.9 10.1 1.0  0.4  5.8  4.7  0.3    5   77.6 9.3  1.0  0.4  7.1  5.4  0.5    RE B        53.2 47.6 9.9  1.3  21.7 7.8  1.8    6   72.3 17.8 2.1  0.7  6.2  2.5  0.4    7   79.2 9.2  1.4  0.3  7.4  3.4  0.3    __________________________________________________________________________

These data show that the present invention provides several advantages.First, the yield of the product alkylated on N⁴ is increased. Second,the yields of products alkylated on N¹ and/or N⁶ are decreased.Therefore, the present invention provides significant improvement inreaction regioselectivity.

EXAMPLE 8 N⁴ -(4-(4-chlorophenyl)benzyl)A82846B Copper Complex

A82846B (0.50 g, 75.6-78.8% potency, 0.24-0.25 mmol) was stirred in 50mL methanol and cupric acetate (53-56 mg, 0.29-0.31 mmol) was addedfollowed by 4'-chloro-4-biphenylcarboxaldehyde (70-73 mg, 0.32-0.34mmol) and sodium cyanoborohydride (20-22 mg, 0.32-0.35 mmol). Thereaction mixture was heated at reflux for 24 hours and cooled to ambienttemperature. The pH was adjusted to 9.0-9.3 by addition of 1 M NaOHsolution. The reaction mixture was concentrated to 10-20 mL on a rotaryevaporator and isopropyl alcohol (13-20 mL) was added dropwise toprecipitate the purple glycopeptide copper complex which was isolated bysuction filtration. Drying in vacuo at 60° C. afforded the glycopeptidecopper complex as a purple powder. After four repetitions of the processthe combined glycopeptide complex was assayed for copper content and wasfound to contain 3.0% copper, confirming a 1:1 copper complex with N⁴-(4-(4-chlorophenyl)benzyl)A82846B.

REFERENCE EXAMPLE C EXAMPLES 9-19

Various copper salts were evaluated in a standardized procedure.

A82846B (1 equivalent as potency adjusted free base) was stirred in 50mL methanol and a divalent metal salt (MX2, 0.63 equivalent) or amonovalent metal salt (MX, 1.25 equivalent) was added followed by4'-chloro-4-biphenylcarboxaldehyde (1.25 equivalent) and sodiumcyanoborohydride (1.25 equivalent). The mixture was heated at reflux for24 hours. After cooling to ambient temperature, an aliquot was removedfor HPLC analysis.

The following HPLC System was used for in situ reaction monitoring andyield calculation: HPLC system Waters 600E with HP3395 integrator andApplied Biosystems 757 detector set at 230 nm, sensitivity 0.1absorption units, 1 sec. filter rise time. Column: DuPont ZorbaxSB-Phenyl, 4.6 mm×25 cm. Eluant A: 10% acetonitrile, 90% buffer (0.2%triethylamine, 0.25% H₃ PO₄). Eluant B: 60% acetonitrile, 40% buffer(0.2% triethylamine, 0.25% H₃ PO₄). Gradient profile at 1 mL/min:initialize 100% A, gradient to 80% A, 20% B over 5 minutes, hold 5minutes, gradient to 100% B over 20 minutes, gradient to 100% A over 5minutes, hold 20 minutes. Sample preparation: 0.5-1.0 g of reactionmixture diluted to 25 mL in acetonitrile-buffer. Hold at ambienttemperature about 30 minutes until the purple color of the coppercomplex is discharged. The desired glycopeptide alkylation productelutes at 16-18 minutes, the starting glycopeptide nucleus at 3-4minutes, the site N⁶ (monosugar) alkylation product at 18-19 minutes,the site N¹ (methyl leucine) alkylation product at 19-21 minutes,dialkylated impurities at 24-26 minutes, and aldehyde at 35-36 minutes.In situ yield is determined by correlation to standards prepared with areference sample of the product.

The results are shown in the following table. Results for alkylatedbyproducts are expressed as percentage relative to the desired N⁴alkylation product.

                                      TABLE II    __________________________________________________________________________                              % Relative to Mono                              on N.sup.4                      Yield        mono                                       mono    Ex. No.         Salt         (%) pH  nucleus                                   on N.sup.6                                       on N.sup.1    __________________________________________________________________________    Ref Ex C         none         63.5                          7.2 25.6 7.8 1.8     9   CuF.sub.2    57.8                          7.2 33.3 6.2 6.1    10   Cu(OH).sub.2 62.0                          7.0 21.3 4.1 1.6    11   Cu(OAc).sub.2                      71.7                          6.4 16.9 3.6 1.8    12   Cu(O.sub.2 CCF.sub.3).sub.2                      64.0                          6.2 17.9 4.0 2.1    13   Cu(cyclohexanebutyrate).sub.2                      69.0                          6.4 15.6 2.3 1.2    14   Cu(2-ethylhexanoate).sub.2                      69.0                          6.5 20.8 3.1 1.4    15   CuCl.sub.2   66.9                          6.2 28.6 4.7 3.3    16   CuBr.sub.2   67.5                          6.1 18.5 3.9 2.4    17   CuCl         67.4                          6.8 23.8 4.1 2.4    18   CuSO.sub.4.5H.sub.2 O                      33.9                          5.8 >100 4.6 1.9    19   CuSO.sub.4   52.1                          6.9 32.2 7.1 8.8    __________________________________________________________________________

The same copper salts were further evaluated for their solubility inmethanol and for the solubility of the starting glycopeptide antibioticin their presence. The procedure was as follows: the copper salt (0.165mmol) was added to 50 mL methanol and stirred at ambient temperature for15 min. Solubility data was recorded as well as the pH. Glycopeptidenucleus (0.55 g, 74.7% potency, 0.41 bg, 0.26 mmol) was added andstirring continued 15 min. Solubility and pH data was recorded.

                  TABLE III    ______________________________________              Salt Solubility in  nucleus solubility    Salt      MeOH         pH     in presence of salt                                            pH    ______________________________________    CuF.sub.2 low, cloudy white                           5.9    slightly, cloudy                                            7.0              soln.               pink    Cu(OH).sub.2              low, cloudy lite blue                           6.2    slightly, cloudy                                            7.0              soln                lite blue    Cu(OAc).sub.2              soluble, clear blue-                           6.5    soluble, clear                                            6.7              green               purple    Cu(O.sub.2 CCF.sub.3).sub.2              soluble, clear lite                           4.4    soluble, clear                                            6.2              blue                purple    Cu(cyclohexane-              faint cloudiness,                           6.0    soluble, clear                                            6.7    butyrate).sub.2              lite blue-green     purple    Cu(2-ethyl-              soluble, clear blue-                           6.5    soluble, clear                                            6.7    hexanoate).sub.2              green               purple    CuCl.sub.2              soluble, clear                           3.2    slightly, cloudy                                            6.6              colorless           purple    CuBr.sub.2              soluble, clear yellow                           2.8    soluble, clear                                            5.9                                  purple    CuSO.sub.4.5H.sub.2 O              soluble, clear                           3.7    slightly, cloudy                                            6.2              colorless           purple    ______________________________________

The foregoing examples illustrate several facets of the presentinvention. First, copper must be supplied to the reaction mixture in aform which is at least partially soluble. Copper salts such as CuF₂ andCu(OH)₂, which are nearly insoluble in methanol, are not effective.Further, the copper salt preferably should allow full solubility of thestarting glycopeptide antibiotic, and ideally at the preferred pH. Thesalts which work the best (Cu(OAc)₂, Cu(cyclohexanebutyrate)₂, andCu(2-ethylhexanoate)₂) afford complete dissolution of nucleus and affordnucleus solutions at about pH 6.7. The salts which afford improvementsover no additive but are not optimal (Cu(O₂ CCF₃)₂, CuCl₂, CuBr₂) eitherafford solubility of nucleus but at less than optimal pH (CuBr₂ andCu(O₂ CCF₃)₂) or are at optimal pH but do not afford complete nucleussolubility (CuCl₂).

In summary, the copper must be in a form which is at least partiallysoluble, and should allow or maintain full solubility of the startingglycopeptide antibiotic at an acceptable pH, typically 6.3-7. Also,these experiments were conducted with suboptimal amounts of the copper;further advantage from the present invention is obtained at highercopper concentration.

REFERENCE EXAMPLE D (NO COPPER) EXAMPLE 20

Two reactions were conducted with the glycopeptide antibiotic A82846A,one without copper (Reference Example D) and one with cupric acetatemonohydrate. The aldehyde was 4'-chloro-4-biphenylcarboxaldehyde. Thereactions were conducted in the essentially same procedures as reportedin the foregoing examples. Results were as set forth in the followingtable:

                  TABLE IV    ______________________________________           HPLC    % Mono-  % Mono-                                   % Di-   % Di-           Area %  alkylated                            alkylated                                   alkylated on                                           alkylated on    Reaction           Yield   on N.sup.6                            on N.sup.1                                   N.sup.4 and N.sup.6                                           N.sup.1 and N.sup.4    ______________________________________    Ref Ex D           52.4    4.7      2.6    15.8    9.0    Example           71.4    1.1      0.9    6.6     6.0    20    ______________________________________

EXAMPLE 21 A82846B Copper Complex

A82846B (3.0 g, 78.7% potency, 2.4 bg, 1.5 mmol) was stirred in 300 mLmethanol at ambient temperature and cupric acetate monohydrate (0.31 g,1.6 mmol) was added. After stirring at ambient temperature for 20minutes, the purple mixture was heated to 35 to 40° C. and stirred anadditional 30 minutes. the solution was concentrated to 45 mL on arotary evaporator and 100 mL or isopropyl alcohol was added dropwiseover 2 hours. The slurry was cooled to 0° C. and filtered. Drying invacuo at 35° C. afforded 2.6 g of the A82846B copper complex as a purplesolid. Mass spectroscopic analysis showed the expected ions for thecomplex, including a series of peaks around 1653, not seen in theanalysis of a reference sample of A82846B, and indicative of theA82846B-copper complex.

Another sample of A82846B copper complex was prepared in like manner andanalyzed by UV-visible spectroscopy, which showed an absorbance maximaat about 540 mm, not seen in the spectra of a reference standard ofA82846B or of cupric acetate and indicative of the A82846B coppercomplex.

We claim:
 1. A method for reductively alkylating a glycopeptideantibiotic comprising an amine-containing saccharide at N⁴ and one ormore other amines, which comprises reacting a soluble copper complex ofthe glycopeptide antibiotic with a ketone or aldehyde in the presence ofa reducing agent which is sodium cyanoborohydride or pyridine•boranecomplex.
 2. A method of claim 1 which is conducted in methanol.
 3. Amethod of claim 2 in which the soluble copper complex of theglycopeptide antibiotic is prepared in situ from the glycopeptideantibiotic and a soluble form of copper.
 4. A method of claim 3 whereinthe glycopeptide antibiotic is a vancomycin type glycopeptideantibiotic.
 5. A method of claim 4 wherein the glycopeptide antibioticis A82846B.
 6. A method of claim 5 wherein the aldehyde is4'-chloro-4-biphenylcarboxaldehyde.